The focus of this proposal is to forge a link between protein- dynamics (backbone and side chain) observable by NMR measurements and stimulated by molecular dynamics calculations and the kinetics of enzyme turnover. Two enzymes will serve as paradigms: the dihydrofolate reductase from Escherichia coli and the metallo-beta-lactamase from Bacteroides fragilis. Both enzymes are medically important: dihydrofolate reductase is a key target for chemotherapeutic agents and the metallo-beta-lactamase is a recently evolved enzyme responsible for the resistance of certain strains of pathogenic bacteria to beta-lactam antibiotics. In Project 3, a kinetic scheme will be obtained for turnover of the metallo-beta- lactamase as well as insights into its mechanism of action. Dependent on the findings from NMR studies of dihydrofolate reductase (Project 1) and metallo-beta-lactamase (Project 2) as to the identity of key amino acid residues with unique dynamic motions, site specific mutagenesis of these residues or regions will be performed and the kinetic schemes for both the metallo-beta-lactamase and the reductase redetermined. Departures both kinetic and mechanistic in mature from the behavior of the wild-type enzymes would constitute strong evidence for the importance of protein dynamics in their respective catalytic cycles.